This invention relates to the preparation and use of nucleic acid fragments or genes encoding monoclonal antibodies immunologically reactive to transit peptides and the use of said genes to create transgenic plants having altered protein levels.
The modification of plants by genetic engineering has been of considerable interest in recent years. To date, several different plant species have been modified by introducing new genes encoding proteins having enzymatic activity. In essence, presence of these enzymes encoded by transgenes results in the production of proteins which can alter existing plant properties and/or produce new and enhanced characteristics. When said proteins are expressed, new or modified enzymatic activities not previously endogenous to the plant can be observed, or levels of proteins found therein can be increased.
Another genetic approach that has been used to modify plant species is down-regulation of a particular plant protein. Down-regulation can result in either partial or complete elimination of a particular enzymatic activity of choice by decreasing overall steady state levels of the protein associated therewith. A common down-regulation approach used to modify a desired plant enzyme level or activity is antisense RNA technology. Antisense RNA results in down-regulation at the RNA translational level. Down-regulation by antisense RNA, as described in U.S. Pat. No. 5,107,065 to Calgene, has been shown effective with a variety of plant genes as described by Shimada et al., (1993) Theor. Appl. Genet. 86:665-672; Kull, et al., (1995) J. Genet. Breed. 49:67-76; Slabas and Elborough, (1997) WO 97/07222 to Zeneca, published Feb. 27, 1997; and Knutzon et al., (1992) Proc. Natl. Acad. Sci USA 89:2624-2628.
Another down regulation approach involves the use of ribozyme technology as described by Hasselhoff and Gerlach, (1988) Nature 342:76-79. Ribozyme technology, like antisense methodologies, also works at the RNA translational level and involves making catalytic RNA molecules which bind to and cleave the mRNA of interest. Ribozymes have recently been demonstrated as an effective method for the down-regulation of plant proteins as described in WO97/10328 to Ribozyme Pharmaceuticals and Dow AgroSciences, LLC, formally DowElanco.
Co-suppression, as described by Seymour et al., (1993, Plant Mol. Biol. 23:1-9) is another approach applicable for down-regulation of plant gene expression. At present, the precise mechanism of down-regulation via co-suppression is not known. However, it has been used extensively to produce transgenic plants having modified gene expression levels as described in Brusslan et al., (1993) Plant Cell 5:667-677; Vaucheret et al., (1995) Mol. Gen. Genet. 248:311-317; and Jorgensen et al., (196) Plant Mol. Biol. 31:957-973.
As disclosed herein, Applicants have invented an alternative approach to down-regulate proteins in plants relying on the use of monoclonal antibodies (MAb) and functional fragments thereof, such as single chain antibodies (SCAb) that specifically recognize and bind transit peptides. As a result, steady-state levels of corresponding passenger proteins can be reduced. The proposed approach is further exemplified, as shown in the non-limiting examples, through down-regulation of maize stearoyl-ACP xcex94-9 desaturase.
In many situations it will be desirable to modify an existing trait of a plant cell rather than introduce a new trait. Thus, one may wish to modify the overall activity levels of a particular enzyme, provide for preferential accumulation of one allele as compared to another, one isozyme as compared to another or the like. In other instances, one may only wish to reduce the amount of expression of a protein encoded by a gene rather than inhibit expression entirely. It is therefore of interest to use the invention disclosed herein which will allow for directed modification of specific proteins in particular plant cells, plant tissues, or plants.
In the present invention, monoclonal antibodies have been generated against transit peptides that direct plant passenger proteins to organelles. Steady state levels of passenger proteins in plant cells can be altered by expressing antibodies which immunologically react to said transit peptides found attached to said passenger proteins in the precursor form.
One aspect of the invention is identification of transit peptides and genes and nucleic acids encoding the same. Information obtained from said genes and nucleic acids can be used to synthesize transit peptides.
Another aspect of the present invention relates to production of hybridoma cells lines producing monoclonal antibodies immunologically reactive to said transit peptides. Said antibodies can exhibit specificity for and high binding affinity to said transit peptides.
Another aspect of the disclosed invention is related to genes and nucleic acid fragments encoding monoclonal antibodies immunologically reactive to plant transit peptides. Said genes are obtained for the heavy chain and light chain portion of said antibodies.
Yet an additional aspect of the present invention relates to modifying genes encoding said monoclonal antibodies thus designing and expressing single chain antibodies (SCAb) derived therefrom. Said SCAbs can exhibit binding specificity and antigen affinity similar to said monoclonal antibodies but are encoded by a single gene.
An additional aspect of the present invention is the production of chimeric genes encoding monoclonal antibodies and single chain antibodies (SCAb) immunologically reactive to said transit peptides in combination with promoter regulatory elements and use of said chimeric genes within a plant cell. Expression of said genes can result in functional antibody proteins which bind to transit peptides and thus lowers steady state levels of passenger proteins in a cell organelle.
Yet an additional aspect of the present invention is transformation of plant species disclosed herein with said chimeric genes.